Bivalent crosslinking of mouse FcγRIII inhibits acLDL endocytosis. (A) Cultured BMM (5 × 105/well) derived from FcγRIIB−/− mice were treated with lipopolysaccharide (LPS; 100 ng/mL) for 12 hours to induce MARCO expression. Cells were preincubated with 2.4G2 F(ab′)2 anti-FcγRII/III (10 μg/mL) or irrelevant rat IgG F(ab′)2 for the indicated times at 37°C, followed by incubation with Alexa Fluor 488–coupled acLDL for 30 minutes to allow endocytosis. Cells were then stained with CD11b-biotin and streptavidin-Alexa 568 on ice for 20 minutes to delineate the cell surface before confocal microscopy. Scale bars: 5 μm. (C) LPS-primed BMMs from FcγRIIB−/− or FcγRIII−/− were treated with irrelevant rat IgG F(ab′)2 (10 μg/mL) or with 2.4G2 F(ab′)2 (10 μg/mL) alone or 2.4G2 F(ab′)2 plus goat anti–rat F(ab′)2 (GAR at 40μg/mL) for 30 minutes at 37°C and acLDL endocytosis was induced as before. Representative merged optical sections are presented showing internalized acDL inside the cells. Scale bars: 5 μm. (B-D) Quantitative analysis of acLDL endocytosis experiments performed in panels A and C, respectively. Green mean fluorescence intensity (MFI) inside each cell was quantified using LSM510 analysis software that analyzes specific pixels in at least 5 confocal microscope fields (*P < .05, **P < .01, ***P < .001; n = 5; nonparametric Mann-Whitney test).
